Energy conversion, utilization and access underlie many of the great challenges of our time, including those associated with sustainability, environmental quality, security, and poverty. New applications of emerging technologies are required to respond to these challenges. Biotechnology, one of the most powerful of the emerging technologies, can give rise to important new energy conversion processes. Plant biomass and derivatives thereof are a resource for the biological conversion of energy to forms useful to humanity.
Among forms of plant biomass, lignocellulosic biomass (“biomass”) is particularly well-suited for energy applications because of its large-scale availability, low cost, and environmentally benign production. In particular, many energy production and utilization cycles based on cellulosic biomass have near-zero greenhouse gas emissions on a life-cycle basis. The primary obstacle impeding the more widespread production of energy from biomass feedstocks is the general absence of low-cost technology for overcoming the recalcitrance of biomass materials to conversion into useful products. Lignocellulosic biomass contains carbohydrate fractions (e.g., cellulose and hemicellulose) including pentose sugars (e.g., xylose and arabinose) that can be converted into ethanol or other products such as lactic acid and acetic acid. In order to convert the lignocellulose fractions, the cellulose, hemicellulose, and pentoses must ultimately be converted into monosaccharides; it is this conversion step that has historically been problematic.
Biomass processing schemes involving enzymatic or microbial hydrolysis commonly involve four biologically mediated transformations: (1) the production of saccharolytic enzymes (cellulases and hemicellulases); (2) the hydrolysis of carbohydrate components present in pretreated biomass to sugars; (3) the fermentation of hexose sugars (e.g., glucose, mannose, and galactose); and (4) the fermentation of pentose sugars (e.g., xylose and arabinose). These four transformations occur in a single step in a process configuration called consolidated bioprocessing (CBP), which is distinguished from other less highly integrated configurations in that it does not involve a dedicated process step for cellulase and/or hemicellulase production.
CBP offers the potential for lower cost and higher efficiency than processes featuring dedicated cellulase production. The benefits result in part from avoided capital costs, substrate and other raw materials, and utilities associated with cellulase production. In addition, several factors support the realization of higher rates of hydrolysis, and hence reduced reactor volume and capital investment using CBP, including enzyme-microbe synergy and the use of thermophilic organisms and/or complexed cellulase systems. Moreover, cellulose-adherent cellulolytic microorganisms are likely to compete successfully for products of cellulose hydrolysis with non- adhered microbes, e.g., contaminants. Successful competition of desirable microbes increases the stability of industrial processes based on microbial cellulose utilization. Progress in developing CBP-enabling microorganisms is being made through two strategies: engineering naturally occurring cellulolytic microorganisms to improve product-related properties, such as yield and titer; and engineering non-cellulolytic organisms that exhibit high product yields and titers to express a heterologous cellulase and hemicellulase system enabling cellulose and hemicellulose utilization.
One way to meet the demand for ethanol production is to convert sugars found in biomass, i.e., materials such as agricultural wastes, corn hulls, corncobs, cellulosic materials, and the like to produce ethanol. Efficient biomass conversion in large-scale industrial applications requires a microorganism that is able to tolerate high concentrations of sugar and ethanol, and which is able to ferment more than one sugar simultaneously.
Pentoses appear in great abundance in nature. As much as 40% of a lignocellulosic material can be comprised of pentoses (Ladisch et ai, “Process considerations in the enzymatic hydrolysis of biomass.” Enz. Microb. Technol., 5: 82-100. (1983)). By fermentation, pentoses can be converted to ethanol which can be used as a liquid fuel or a chemical feedstock. Although many microorganisms have the ability to ferment simple hexose sugars, the pentose sugars, xylose and arabinose, are among the most difficult sugars in biomass to metabolize. Some microorganisms can ferment pentoses to ethanol and other co-products, and microorganisms with improved ethanol production from pentose sugars have been genetically engineered. However, many of these studies have been conducted in bacteria that are sensitive to low pH and high concentrations of ethanol. Therefore, their use in fermentations is associated with undesired co-product formation, and the level of ethanol they are capable of producing remains low.
Bakers' yeast (Saccharomyces cerevisiae) is the preferred microorganism for the production of ethanol (Hahn-Hagerdal, B., et ah, Adv. Biochem. Eng. Biotechnol. 73, 53-84 (2001)). Attributes in favor of this microbe are (i) high productivity at close to theoretical yields (0.51 g ethanol produced/g glucose used), (ii) high osmo- and ethanol tolerance, (iii) natural robustness in industrial processes, also (iv) being generally regarded as safe (GRAS) due to its long association with wine and bread making, and beer brewing. Furthermore, S. cerevisiae exhibits tolerance to inhibitors commonly found in hydrolysates resulting from biomass pretreatment. However, S. cerevisiae does not naturally break down components of cellulose, nor does it efficiently use pentose sugars.
Progress has been made in engineering S. cerevisiae to express heterologous enzymes that enable it to break down cellulose. (See e.g. U.S. application Ser. No. 13/130,549 and PCT/US2011/039192, incorporated herein by reference in their entirety). However, utilization of arabinose for industrial ethanologenic fermentation has not been demonstrated in yeast. In addition, there is a need for an ethanologenic organism capable of efficiently utilizing arabinose and xylose that is also capable of breaking down cellulose. The highest products yields are obtained when all the cellulose and hemicellulose are broken down into monomer sugars and fermented into the desired product.
Therefore, there is a need in the art for an ethanologenic organism capable of fermenting pentose sugars in quantities sufficient for commercial applicability. There is also a need to combine efficient pentose utilization with cellulose digestion in order to maximize the efficiency of cellulosic feedstock use and to generate the highest yield of product.